It is proposed to continue the present work of years 18-20 of separating mycobacterial antigens in culture filtrate or cellular extracts, by new methodology, in view of the importance of tuberculin hypersensitivity in delayed hypersensitivity. We have already shown that at least 5, surely more, delayed-type hypersensitivities arise to mycobacterial antigens when the body encounters the mycobacterial cell. Clones of cells which are committed to recognizing single antigens are present but are not called into play when tuberculin PPD is the sole test material for MIF synthesis by human or animal lymphocytes. Native antigenic proteins permissible in in vitro testing are undesirable for human skin testing. There is one antigen ("Antigen-3" or "AG-3") present in all 14 commercial PPD preparations tested; it is relatively stable to heating at 100 degrees C. We desire to separate this Ag-3 in native form and to study any changes which arise during heating. Separated antigens would lead to new studies. An established methodology would be applicable to separation of antigens of atypical mycobacteria and of M. leprae--tasks for others to pursue. The basic concept of our study is to make biochemical simplifications by appropriate fractionation steps before finally resorting to polyacrylamide gel electrophoresis (PAGE) to separate small assortments of antigens. Steps used include: frationation by brief exposure to acetate at pH 4.5; sieving on Sephadex G75 superfine; a narrow Fraction II is insolubilized at 0.9 saturated ammonium sulfate ("0.9 SAS") in the cold and then solubilized stepwise by successively lowered concentrations; assignment of Rf values to proteins of interest in PAGE by penetrance into the gel vs that of a Myoglobin Standard; and finally preparative PAGE based on the Rf values. At any step, we can determine the antigens in fractions and roughly the amounts by a special procedure of passive cutaneous anaphylaxis with use of 10 specific guinea pig IgG1 antisera. By these means, 2 of the antigens have been separated on a small scale.